The human ribosomal gene is a 44 kb unit that forms a tandemly repeated complex at the secondary constrictions of the five acrocentric chromosomes. The gene is composed of a 31 kb intergenic spacer (IGS) and a 13.3 Kb region encoding the primary transcript that is processed to the three mature (18S, 5.8S and 28S) ribosomal RNAs. This proposal seeks to determine the primary sequence and to analyze the organization of the components of this large gene with special regard to function, normal variation, evolution and to their location in the nucleolus. Sequences immediately upstream of the promoter will be tested as to whether they are transcribed and involved in the regulation of the major transcription unit OS is the case for other organisms. The extent and type of variation of expressed rRNA will be determined by sequencing cDNAs generated from various parts of the primary transcript. The molecular basis for a large size variation within the IGS will be determined. Genomic clones from human land the great apes will be analyzed to trace the evolution of the IGS. The exact nucleotide sequence at the distal junction of the tandem array will be determined and compared for each acrocentric chromosome of at least two individuals from different populations. An in vitro replication system that faithfully represents the in vivo situation will be used to characterize the sequence requirements for initiating DNA replication within the ribosomal repeat. The precise locations of various rDNA segments and of other acrocentric chromosome sequences during the cell cycle and in the nucleolus will be determined by in situ hybridization followed by both light and electron microscopy. The goal of gathering information and obtaining a thorough understanding of the human ribosomal gene is important because the gene and/or its RNA has a central role in events such as nondisjunction, nucleolus formation, antibiotic susceptibility, protein synthesis and the regulation of cell growth.